Dystonia is characterized by involuntary muscle contractions that cause twisting movements and postures. Although basal ganglia dysfunction is consistently implicated across many forms of dystonia, the underlying pathophysiology is unknown. The major input structure of the basal ganglia is the striatum. The vast majority of neurons in the striatum are GABAergic spiny projection neurons (SPNs). Direct pathway SPNs (dSPNs) project to the internal pallidum (GPi) to promote movement. Indirect pathway SPNs (iSPNs) project to the external pallidum (GPe) to inhibit movement. Although dSPNs and dSPNs are largely segregated into separate pathways, they act in concert and within ensembles of coactivity to select and refine movements. In dystonia patients, this coordinated activity appears to be disrupted as microelectrode recordings in dystonia patients reveal abnormal activity in both GPi and GPe. Although these results implicate abnormal activity in both dSPNs and iSPNs, the abnormal patterns of striatal activity that mediate dystonia are unknown. Several challenges have stymied our ability to understand the abnormal neural code underlying dystonia. First, the information obtained from microelectrode recording in patients is, by necessity, quite limited. Second, because dystonia is induced by movement, recordings must be made in awake unrestrained subjects to obtain authentic results. Third, to meaningfully understand striatal pathophysiology, it is necessary to distinguish between dSPNs and iSPNs, which is challenging to accomplish using microelectrode recordings in vivo because these two distinct cells types are intermingled within the striatum. With the recent development of lightweight miniature fluorescence microscopes that can be used to image the activity of genetically-identified SPNs in freely moving mice, we can overcome these obstacles to understand the physiological substrates of dystonia. For the first experiments to reveal abnormal patterns of striatal activity in dystonia, we will use a knockin mouse model of DOPA-responsive dystonia (DRD) in which the striatum is known to play a central role in mediating the dystonia. In fact, both dSPN and iSPN signaling is disrupted in these mice. Thus, we now have an unprecedented opportunity to elucidate the neural code of dystonia for the first time. The Specific Aim is to identify abnormal patterns of striatal activity in dystonia. We will test the hypothesis that dystonia is mediated by abnormal neuronal activity and degraded ensemble activity of dSPNs and iSPNs by performing in vivo Ca2+ imaging in freely moving normal or DRD mice that selectively express the GCaMP6f calcium indicator in either dSPNs or iSPNs. Abnormal firing patterns including the degree of spatial clustering of coactive neurons will be assessed using both well-established approaches and a novel analysis of population dynamics using a machine learning algorithm that we recently developed to identify and predict dystonia-specific neuronal ensemble firing patterns.